Leptin, principally produced in adipose tissue, has a central physiologic role in providing information on energy stores and energy balance to brain centers that regulate appetite, energy expenditure and neuroendocrine function. One of the actions of leptin is to activate the Hypothalamic-Pituitary-Thyroid (HPT) axis through the thyrotropin-releasing hormone (TRH) peptide thereby increasing energy expenditure. During the last three years of funding, we were able to provide strong evidence for the existence of a direct (paraventricular nucleus, PVN) and an indirect (arcuate nucleus, ARC) pathway of leptin action on the regulation of the TRH prohormone biosynthesis. We demonstrated that leptin could directly regulate proTRH biosynthesis and TRH secretion in cultured hypothalamic neurons independently of the melanocortin (alpha-melanocyte-stimulating-hormone, alpha-MSH) input. We also observed the colocalization of the leptin receptor (ObRb) with proTRH in neurons of the PVN, strongly suggesting direct effects of leptin on TRH neurons. This hypothesis was further supported by the demonstration of ObRb mRNA expression within the PVN, and by showing that TRH neurons express SOCS-3 mRNA (which is the sensitive marker of direct leptin action) after leptin administration to rats. Recently, we demonstrated for the first time activation of STATS in TRH neurons by leptin in vivo, providing further evidence of a potential key role of the STAT3 transcription factor to regulate the activity of the proTRH promoter by leptin. We also recently demonstrated that the increase in TRH biosynthesis due to leptin action was associated with an up-regulation of the processing enzymes PCI and PC2, involved in the maturation of TRH and other proTRH peptides. Aim #1 We will test the hypothesis that there are two different or overlapping groups of PVN-TRH neurons carrying the ObRb (direct pathway) and the MC4R (indirect pathway) that are targeted by leptin and alpha-MSH to regulate the energy balance by increasing energy expenditure through the HPT axis, and potentially inhibiting food intake by sending synapses to the LH and DMH. Aim #2 We will test the hypothesis that leptin regulates the HPT axis primarily through the direct pathway. AIM #3 A) Because we found that leptin regulates proTRH and coordinates its processing by also up-regulating PCI and PC2 biosynthesis, we hypothesize that leptin might also regulate the other enzymes, co-chaperones, and inhibitors necessary for full prohormone processing and maturation. B) Since the melanocortin system represents the indirect pathway of action on TRH neurons, we will also determine whether alpha- MSH affects the biosynthesis, maturation and activity of PCI, PC2, proSAAS, 7B2 and CPE.